Imaging of the Lactate/Pyruvate Ratio Using a Genetically Encoded Förster Resonance Energy Transfer Indicator

Area of Science:

• Biochemistry
• Cell Biology
• Metabolic Engineering

Background:

• The lactate/pyruvate ratio is a key indicator of cellular metabolic state, reflecting the balance between glycolysis and mitochondrial respiration.
• Existing methods lack the spatial and temporal resolution required for real-time monitoring of this ratio in living systems.

Purpose of the Study:

• To develop a novel, genetically encoded indicator for real-time, high-resolution measurement of the lactate/pyruvate ratio in living cells.
• To characterize the properties and validate the performance of this new indicator.

Main Methods:

• Utilized a Förster resonance energy transfer (FRET)-based screening strategy to identify a suitable sensor protein.
• Engineered a FRET-based genetically encoded indicator, named Lapronic, using the LutR transcriptional factor.
• Validated Lapronic's sensitivity, specificity, and stability using isothermal titration calorimetry and expression in HEK 293 cells.

Main Results:

• Identified LutR as an endogenous sensor of the lactate/pyruvate ratio.
• Developed Lapronic, a FRET-based indicator demonstrating sensitivity to lactate and pyruvate.
• Lapronic exhibited stability at physiological pH and temperature, and specificity against related molecules and cofactors.
• Successfully calibrated Lapronic for quantitative lactate/pyruvate ratio assessment in mammalian cells.

Conclusions:

• Lapronic provides a robust and sensitive method for measuring the lactate/pyruvate ratio in real-time within living cells.
• This indicator is suitable for cytosolic and mitochondrial localization, offering versatile applications in metabolic research.
• Lapronic is expected to be valuable for both basic and applied research in cellular metabolism.